Pathologic calcium phosphate deposition in the blood vessels, myocardium and cardiac valves (also called cardiovascular calcification) is a common complication associated with diabetes, hypercholesterolemia, chronic renal insufficiency, osteoporosis, atherosclerosis, and ageing. Despite the life threatening significance of vascular calcification, its underlying mechanisms have not been clearly defined. Extracellular enzyme transglutaminase 2 (TG2) appears to be a critical regulator of calcification in vascular smooth muscle cells (VSMCs), however the mechanism by which TG2 promotes this pathology remains unknown. Unraveling this mechanism is essential for identification of novel therapies to prevent and/or treat vascular calcification. We have found that extracellular TG2, which often accumulates at the sites of vascular calcification, significantly enhances calcification in cultured VSMCs and promotes their osteoblast-like transformation. At the same time, TG2 activates 2-catenin in VSMCs and binds to the Low-density lipoprotein receptor-related proteins 5/6 (LRP5/6) - the key receptors of the canonical Wnt/2-catenin signaling. Moreover, LRP5 is modified by the TG2-mediated cross-linking. Our central hypothesis is that vascular calcification is promoted by the TG2-LRP5/6-mediated activation of the 2-catenin signaling pathway. This hypothesis will be tested by pursuing three Specific Aims. In Aim1, the role for LRP5/6 receptors in TG2- induced vascular calcification will be analyzed in VSMC cultures and aortic rings with down-regulated levels of both LRP5 and LRP6. In addition, the mechanism of the TG2-LRP5/6 binding will be analyzed in order to provide approach for interfering with this binding and thus blocking VSMC calcification. Under the second Aim, the requirement for the cross-linking activity of TG2 in vascular calcification in vivo, and in theTG2- induced activation of the LRP5/6 receptors will be determined using the catalytically inactive TG2 mutant protein and a specific inhibitor of the transamidating activity of TG2. Under the third Aim, the role of the canonical 2-catenin signaling pathway in the TG-induced mineralization of VSMCs will be addressed by disrupting the 2-catenin signaling using genetic approach and overexpression of negative regulators. Further, the efficacy of pharmacological inhibition of the 2-catenin signaling in regulation of vascular calcification will be determined in vitro, ex vivo, and in vivo. Our studies will advance scientific knowledge about alternative mediators of 2-catenin activation in vessel wall, as well as transglutaminase function. Defining these pathways will lead to new mechanistic insights that could translate into novel therapeutic strategies for vascular calcification and thus improve clinical treatment of this pathology. Additionally, our studies will broadly impact several medical fields in which both 2-catenin and transglutaminases have been implicated, ranging from cardiovascular disease to cancer and bone remodeling. PUBLIC HEALTH RELEVANCE: We propose to study the molecular mechanisms of vascular calcification induced by a novel regulator-extracellular transglutaminase. Our findings will illuminate the role of this regulator and of the 2-catenin pathway in this pathology, thus providing a scientific basis for development of therapeutic approaches in prevention and/or treatment of vascular calcification. In addition, the proposed work will impact research in multiple areas ranging from cardiovascular disease to cancer and bone remodeling, by improving understanding of the regulation of 2-catenin signaling and of the biological roles of transglutaminases.